Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers

ABSTRACT

The present invention provides a new method for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating gastointestinal cancers including small intestine, colon and stomach cancer.

This patent application is the U.S. National Stage of InternationalApplication PCT/US99/22725, filed Sep. 30, 1999 which claims the benefitof priority from U.S. Provisional Application Ser. No. 60/102,879, filedOct. 2, 1998.

FIELD OF THE INVENTION

This invention relates, in part, to newly developed assays fordetecting, diagnosing, monitoring, staging prognosticating, imaging andtreating cancers, particularly gastrointestinal cancers including cancerof the stomach, small intestine and colon.

BACKGROUND OF THE INVENTION

Cancer of the colon is the second most frequently diagnosed malignancyin the United States, as well as the second most common cause of cancerdeath. Colon cancer is a highly treatable and often curable disease whenlocalized to the bowel. Surgery is the primary treatment and results incure in approximately 50% of patients. However, recurrence andmetastases following surgery is a major problem and often is theultimate cause of death.

Due to its proximity, cancer of the colon often metastasizes to thesmall intestine. The prognosis of the cancer spreading to the smallintestine is related to the degree of penetration of the tumor throughthe bowel wall and the presence or absence of nodal involvement. Thesetwo characteristics form the basis for all staging systems developed forcolon cancer. Various characteristics also assist in prognosticatingcolon cancer and its spread to the small intestines. For example, bowelobstruction and bowel perforation are indicators of poor prognosis.Elevated pretreatment serum levels of carcinoembryonic antigen (CEA) andof carbohydrate antigen 19-9 (CA 19-9) also have a negative prognosticsignificance. However, age greater than 70 years at presentation is nota contraindication to standard therapies; acceptable morbidity andmortality, as well as long-term survival, are achieved in this patientpopulation.

Cancer cells can also originate in the small intestine. However, this isa much rarer type of cancer.

Symptoms of cancer of the small intestine typically include pain orcramps in the middle of the abdomen, weight loss without dieting, a lumpin the abdomen or blood in the stool.

Cancer of the stomach, also referred to as gastric cancer, alsofrequently metastasizes to the small intestine due to its proximity.This cancer is often difficult to diagnose in early stages and can be inthe stomach for a long time, growing to a large size before symptomsarise. In the early stages of cancer of the stomach, an individual mayexperience indigestion and stomach discomfort, a bloated feeling aftereating, mild nausea, loss of appetite or heartburn. In more advancedstages of stomach cancer, there may be blood in the stool, vomiting,weight loss or more severe pain.

Because of the frequency of these types of cancer (approximately 160,000new cases of colon and rectal cancer per year alone), the identificationof high-risk groups, the demonstrated slow growth of primary lesions andthe better survival of early-stage lesions, screening forgastrointestinal cancers should be a part of routine care for all adultsstarting at age 50, especially those with first-degree relatives withcolorectal cancer.

Procedures used for detecting, diagnosing, monitoring, staging, andprognosticating cancer of the colon, small intestine or stomach are ofcritical importance to the outcome of the patient. Patients diagnosedwith early stage cancer generally have a much greater five-year survivalrate as compared to the survival rate for patients diagnosed withdistant metastasized cancers. New diagnostic methods which are moresensitive and specific for detecting early cancer of the stomach, smallintestine and colon are clearly needed.

Patients with gastrointestinal cancers are closely monitored followinginitial therapy and during adjuvant therapy to determine response totherapy and to detect persistent or recurrent disease of metastasis.There is clearly a need for a cancer marker which is more sensitive andspecific in detecting recurrence of these types of cancer.

Another important step in managing gastrointestinal cancers is todetermine the stage of the patient's disease. Stage determination haspotential prognostic value and provides criteria for designing optimaltherapy. Generally, pathological staging of cancer is preferable overclinical staging because the former gives a more accurate prognosis.However, clinical staging would be preferred were it at least asaccurate as pathological staging because it does not depend on aninvasive procedure to obtain tissue for pathological evaluation. Stagingof gastrointestinal cancers would be improved by identifying new markersin cells, tissues, or bodily fluids which could differentiate betweendifferent stages of invasion.

Thirteen colon specific genes and naturally occurring variants thereof,referred to as CSG1-13, are disclosed in U.S. Pat. No. 5,733,748 and WO96/39541 for use as diagnostic markers in colon cancer. Some of thesegenes and polypeptides encoded thereby are also taught to be useful indetermining if the colon cancer has metastasized.

U.S. Pat. No. 5,861,494, which issued January 19, 1999, also discloses agene and polypeptide encoded thereby for use as a diagnostic marker forcolon cancer and as an agent for determining if the colon cancer hasmetastasized. This gene and the polypeptide encoded thereby are similarin sequence to the cancer specific gene referred to herein as CC2.

It has now been found that CC2 is a useful diagnostic and metastaticmarker not only for colon cancer but also for cancer of the stomach andsmall intestine. Thus, in the present invention, methods are providedfor detecting, diagnosing, monitoring, staging, prognosticating, imagingand treating gastrointestinal cancers including cancer of the stomach,small intestine and colon via the cancer specific gene referred toherein as CC2. CC2 refers, among other things, to native proteinexpressed by the gene comprising the polynucleotide sequence of SEQ IDNO:1. The amino acid sequence of a polypeptide encoded by SEQ ID NO:1 isdepicted herein as SEQ ID NO:2. In the alternative, what is meant by CC2as used herein, means the native mRNA encoded by the gene comprising thepolynucleotide sequence of SEQ ID NO:1 or levels of the gene comprisingthe polynucleotide sequence of SEQ ID NO:1.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide a method for diagnosing the presence of a gastrointestinalcancer by analyzing for changes in levels of CC2 in cells, tissues orbodily fluids compared with levels of CC2 in preferably the same cells,tissues, or bodily fluid type of a normal human control, wherein achange in levels of CC2 in the patient versus the normal human controlis associated with a gastrointestinal cancer.

Further provided is a method of diagnosing metastatic cancer in apatient having a gastrointestinal cancer which is not known to havemetastasized by identifying a human patient suspected of having agastrointestinal cancer that has metastasized; analyzing a sample ofcells, tissues, or bodily fluid from such patient for CC2; comparing theCC2 levels in such cells, tissues, or bodily fluid with levels of CC2 inpreferably the same cells, tissues, or bodily fluid type of a normalhuman control, wherein an increase in CC2 levels in the patient versusthe normal human control is associated with a gastrointestinal cancerwhich has metastasized.

Also provided by the invention is a method of staging a gastrointestinalcancer in a human which has such cancer by identifying a human patienthaving such cancer; analyzing a sample of cells, tissues, or bodilyfluid from such patient for CC2; comparing CC2 levels in such cells,tissues, or bodily fluid with levels of CC2 in preferably the samecells, tissues, or bodily fluid type of a normal human control sample,wherein an increase in CC2 levels in the patient versus the normal humancontrol is associated with a cancer which is progressing and a decreasein the levels of CC2 is associated with a cancer which is regressing orin remission.

Further provided is a method of monitoring a gastrointestinal cancer ina human having such cancer for the onset of metastasis. The methodcomprises identifying a human patient having such cancer that is notknown to have metastasized; periodically analyzing a sample of cells,tissues, or bodily fluid from such patient for CC2; comparing the CC2levels in such cells, tissue, or bodily fluid with levels of CC2 inpreferably the same cells, tissues, or bodily fluid type of a normalhuman control sample, wherein an increase in CC2 levels in the patientversus the normal human control is associated with a cancer which hasmetastasized.

Further provided is a method of monitoring the change in stage of agastrointestinal cancer in a human having such cancer by looking atlevels of CC2 in a human having such cancer. The method comprisesidentifying a human patient having such cancer; periodically analyzing asample of cells, tissues, or bodily fluid from such patient for CC2;comparing the CC2 levels in such cells, tissue, or bodily fluid withlevels of CC2 in preferably the same cells, tissues, or bodily fluidtype of a normal human control sample, wherein an increase in CC2 levelsin the patient versus the normal human control is associated with acancer which is progressing and a decrease in the levels of CC2 isassociated with a cancer which is regressing or in remission.

Further provided are antibodies targeted against CC2 or fragments ofsuch antibodies which can be used to detect or image localization of CC2in a patient for the purpose of detecting or diagnosing a disease orcondition. Such antibodies can be polyclonal, monoclonal, or omniclonalor prepared by molecular biology techniques. The term “antibody”, asused herein and throughout the instant specification is also meant toinclude aptamers and single-stranded oligonucleotides such as thosederived from an in vitro evolution protocol referred to as SELEX andwell known to those skilled in the art. Antibodies can be labeled with avariety of detectable labels including, but not limited to,radioisotopes and paramagnetic metals. These antibodies or fragmentsthereof can also be used as therapeutic agents in the treatment ofdiseases characterized by expression of CC2. In therapeuticapplications, the antibody can be used without or with derivatization toa cytotoxic agent such as a radioisotope, enzyme, toxin, drug or aprodrug.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to diagnostic assays and methods, bothquantitative and qualitative for detecting, diagnosing, monitoring,staging and prognosticating cancers by comparing levels of CC2 withthose of CC2 in a normal human control. What is meant by levels of CC2as used herein, means levels of the native protein expressed by the genecomprising the polynucleotide sequence of SEQ ID NO:1. The amino acidsequence of a polypeptide encoded by SEQ ID NO:1 is depicted herein asSEQ ID NO:2. In the alternative, what is meant by levels of CC2 as usedherein, means levels of the native mRNA encoded by the gene comprisingthe polynucleotide sequence of SEQ ID NO:1 or levels of the genecomprising the polynucleotide sequence of SEQ ID NO:1. Such levels arepreferably measured in at least one of cells, tissues and/or bodilyfluids, including determination of normal and abnormal levels. Thus, forinstance, a diagnostic assay in accordance with the invention fordiagnosing overexpression of CC2 protein compared to normal controlbodily fluids, cells, or tissue samples may be used to diagnose thepresence of cancers, and in particular gastrointestinal cancers. Bygastrointestinal cancers it is meant to include stomach cancer, cancerof the small intestine, and colon cancer.

All the methods of the present invention may optionally includemeasuring levels of other cancer markers as well as CC2. Other cancermarkers, in addition to CC2, useful in the present invention will dependon the cancer being tested and are known to those of skill in the art.

Diagnostic Assays

The present invention provides methods for diagnosing the presence of agastrointestinal cancer by analyzing for changes in levels of CC2 incells, tissues or bodily fluids compared with levels of CC2 in cells,tissues or bodily fluids of preferably the same type from a normal humancontrol, wherein a change in levels of CC2 in the patient versus thenormal human control is associated with the presence of agastrointestinal cancer.

Without limiting the instant invention, typically, for a quantitativediagnostic assay a positive result indicating the patient being testedhas cancer is one in which cells, tissues or bodily fluid levels of thecancer marker, such as CC2, are at least two times higher, and mostpreferably are at least five times higher, than in preferably the samecells, tissues or bodily fluid of a normal human control.

The present invention also provides a method of diagnosing the onset ofmetastatic gastrointestinal cancers in a patient having agastrointestinal cancer which has not yet metastasized. In the method ofthe present invention, a human cancer patient suspected of having agastrointestinal cancer which may have metastasized (but which was notpreviously known to have metastasized) is identified. This isaccomplished by a variety of means known to those of skill in the art.

In the present invention, determining the presence of CC2 levels incells, tissues or bodily fluid, is particularly useful fordiscriminating between gastrointestinal cancers which have notmetastasized and gastrointestinal cancers which have metastasized.Existing techniques have difficulty discriminating betweengastrointestinal cancers which have metastasized and gastrointestinalcancers which have not metastasized. However, proper treatment selectionis often dependent upon such knowledge.

In the present invention, the cancer marker level measured in cells,tissues or bodily fluid of a human patient is CC2. The measured CC2level in the human patient is compared with levels of CC2 in preferablythe same cells, tissue or bodily fluid type of a normal human control.That is, if the cancer marker being observed is CC2 in serum, this levelis preferably compared with the level of CC2 in serum of a normal humancontrol. An increase in the CC2 in the patient versus the normal humancontrol is associated with a gastrointestinal cancer which hasmetastasized.

Without limiting the instant invention, typically, for a quantitativediagnostic assay a positive result indicating the cancer in the patientbeing tested or monitored has metastasized is one in which cells,tissues or bodily fluid levels of the cancer marker, such as CC2, are atleast two times higher, and most preferably are at least five timeshigher, than in preferably the same cells, tissues or bodily fluid of anormal patient.

Normal human control as used herein includes a human patient withoutcancer and/or non cancerous samples from the patient; in the methods fordiagnosing or monitoring for metastasis, normal human control maypreferably also include samples from a human patient that is determinedby reliable methods to have a gastrointestinal cancer which has notmetastasized.

Staging

The invention also provides a method of staging gastrointestinal cancersin a human patient. The method comprises identifying a human patienthaving such cancer and analyzing a sample of cells, tissues or bodilyfluid from such human patient for CC2. In this method CC2 levels in suchcells, tissues or bodily fluid are then compared with levels of CC2 inpreferably the same cells, tissues or bodily fluid type of a normalhuman control sample, wherein an increase in CC2 levels in the humanpatient versus the normal human control is associated with a cancerwhich is progressing and a decrease in the levels of CC2 is associatedwith a cancer which is regressing or in remission.

Monitoring

Further provided is a method of monitoring gastrointestinal cancers in ahuman having such cancer for the onset of metastasis. The methodcomprises identifying a human patient having such cancer that is notknown to have metastasized; periodically analyzing a sample of cells,tissues or bodily fluid from such human patient for CC2; comparing theCC2 levels in such cells, tissues or bodily fluid with levels of CC2 inpreferably the same cells, tissues or bodily fluid type of a normalhuman control, wherein an increase in CC2 levels in the human patientversus the normal human control is associated with a cancer which hasmetastasized.

Further provided by this invention is a method of monitoring the changein stage of gastrointestinal cancers in a human having such cancer. Themethod comprises identifying a human patient having such cancer;periodically analyzing a sample of cells, tissues or bodily fluid fromsuch human patient for CC2; and comparing the CC2 levels in such cells,tissues or bodily fluid with levels of CC2 in preferably the same cells,tissues or bodily fluid type of a normal human control, wherein anincrease in CC2 levels in the human patient versus the normal humancontrol is associated with a cancer which is progressing in stage and adecrease in the levels of CC2 is associated with a cancer which isregressing in stage or in remission.

Monitoring such patient for onset of metastasis is periodic andpreferably done on a quarterly basis. However, this may be more or lessfrequent depending on the cancer, the particular patient, and the stageof the cancer.

Assay Techniques

Assay techniques that can be used to determine levels of gene expression(including protein levels), such as CC2 of the present invention, in asample derived from a patient are well known to those of skill in theart. Such assay methods include, without limitation, radioimmunoassays,reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays,in situ hybridization assays, competitive-binding assays, Western Blotanalyses, ELISA assays and proteomic approaches: two-dimensional gelelectrophoresis (2D electrophoresis) and non-gel based approaches suchas mass spectrometry or protein interaction profiling. Among these,ELISAs are frequently preferred to diagnose a gene's expressed proteinin biological fluids.

An ELISA assay initially comprises preparing an antibody, if not readilyavailable from a commercial source, specific to CC2, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds specifically to CC2. The reporter antibody isattached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, for example horseradish peroxidase enzyme or alkalinephosphatase.

To carry out the ELISA, antibody specific to CC2 is incubated on a solidsupport, e.g. a polystyrene dish, that binds the antibody. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein such as bovine serum albumin. Next, the sample tobe analyzed is incubated in the dish, during which time CC2 binds to thespecific antibody attached to the polystyrene dish. Unbound sample iswashed out with buffer. A reporter antibody specifically directed to CC2and linked to horseradish peroxidase is placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound toCC2. Unattached reporter antibody is then washed out. Reagents forperoxidase activity, including a calorimetric substrate are then addedto the dish. Immobilized peroxidase, linked to CC2 antibodies, producesa colored reaction product. The amount of color developed in a giventime period is proportional to the amount of CC2 protein present in thesample. Quantitative results typically are obtained by reference to astandard curve.

A competition assay can also be employed wherein antibodies specific toCC2 are attached to a solid support and labeled CC2 and a sample derivedfrom the host are passed over the solid support. The amount of labeldetected which is attached to the solid support can be correlated to aquantity of CC2 in the sample.

Nucleic acid methods can also be used to detect CC2 mRNA as a marker forgastrointestinal cancers. Polymerase chain reaction (PCR) and othernucleic acid methods, such as ligase chain reaction (LCR) and nucleicacid sequence based amplification (NASABA), can be used to detectmalignant cells for diagnosis and monitoring of various malignancies.For example, reverse-transcriptase PCR (RT-PCR) is a powerful techniquewhich can be used to detect the presence of a specific mRNA populationin a complex mixture of thousands of other mRNA species. In RT-PCR, anmRNA species is first reverse transcribed to complementary DNA (CDNA)with use of the enzyme reverse transcriptase; the CDNA is then amplifiedas in a standard PCR reaction. RT-PCR can thus reveal by amplificationthe presence of a single species of mRNA. Accordingly, if the mRNA ishighly specific for the cell that produces it, RT-PCR can be used toidentify the presence of a specific type of cell.

Hybridization to clones or oligonucleotides arrayed on a solid support(i.e. gridding) can be used to detect both the expression of andquantitate the level of expression of a gene. In this approach, a cDNAencoding the CC2 gene is fixed to a substrate. The substrate may be ofany suitable type including but not limited to glass, nitrocellulose,nylon or plastic. At least a portion of the DNA encoding the CC2 gene isattached to the substrate and then incubated with the analyte, which maybe RNA or a complementary DNA (cDNA) copy of the RNA, isolated from thetissue of interest. Hybridization between the substrate bound DNA andthe analyte can be detected and quantitated by several means includingbut not limited to radioactive labeling or fluorescence labeling of theanalyte or a secondary molecule designed to detect the hybrid.Quantitation of the level of gene expression can be done by comparisonof the intensity of the signal from the analyte compared with thatdetermined from known standards. The standards can be obtained by invitro transcription of the target gene, quantitating the yield, and thenusing that material to generate a standard curve.

Of the proteomic approaches, 2D electrophoresis is a technique wellknown to those in the art. Isolation of individual proteins from asample such as serum is accomplished using sequential separation ofproteins by different characteristics usually on polyacrylamide gels.First, proteins are separated by size using an electric current. Thecurrent acts uniformly on all proteins, so smaller proteins move fartheron the gel than larger proteins. The second dimension applies a currentperpendicular to the first and separates proteins not on the basis ofsize but on the specific electric charge carried by each protein. Sinceno two proteins with different sequences are identical on the basis ofboth size and charge, the result of a 2D separation is a square gel inwhich each protein occupies a unique spot. Analysis of the spots withchemical or antibody probes, or subsequent protein microsequencing canreveal the relative abundance of a given protein and the identity of theproteins in the sample.

The above tests can be carried out on samples derived from a variety ofcells, bodily fluids and/or tissue extracts (homogenates or solubilizedtissue) obtained from a patient including those from tissue biopsies andautopsy material. Bodily fluids useful in the present invention includeblood, urine, saliva or any other bodily secretion or derivativethereof. Blood can include whole blood, plasma, serum or any derivativeof blood.

In Vivo Antibody Use

Antibodies which specifically bind to CC2 can also be used in vivo inpatients suspected of suffering from gastrointestinal cancers includingstomach cancer, cancer of the small intestine, and colon cancer.Specifically, antibodies which specifically bind a CC2 can be injectedinto a patient suspected of having a gastrointestinal cancer fordiagnostic and/or therapeutic purposes. The preparation and use ofantibodies for in vivo diagnosis is well known in the art. For example,antibody-chelators labeled with Indium-ill have been described for usein the radioimmunoscintographic imaging of carcinoembryonic antigenexpressing tumors (Sumerdon et al. Nucl. Med. Biol. 1990 17:247-254). Inparticular, these antibody-chelators have been used in detecting tumorsin patients suspected of having recurrent colorectal cancer (Griffin etal. J. Clin. Onc. 1991 9:631-640). Antibodies with paramagnetic ions aslabels for use in magnetic resonance imaging have also been described(Lauffer, R. B. Magnetic Resonance in Medicine 1991 22:339-342).Antibodies directed against CC2 can be used in a similar manner. Labeledantibodies which specifically bind CC2 can be injected into patientssuspected of having a gastrointestinal cancer for the purpose ofdiagnosing or staging of the disease status of the patient. The labelused will be selected in accordance with the imaging modality to beused. For example, radioactive labels such as Indium-111, Technetium-99mor Iodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT) . Positron emitting labels such asFluorine-19 can be used in positron emission tomography. Paramagneticions such as Gadlinium (III) or Manganese (II) can be used in magneticresonance imaging (MRI). Localization of the label permits determinationof the spread of the cancer. The amount of label within an organ ortissue also allows determination of the presence or absence of cancer inthat organ or tissue.

For patients diagnosed with a gastrointestinal cancer, injection of anantibody which specifically binds CC2 can also have a therapeuticbenefit. The antibody may exert its therapeutic effect alone.Alternatively, the antibody may be conjugated to a cytotoxic agent suchas a drug, toxin or radionuclide to enhance its therapeutic effect. Drugmonoclonal antibodies have been described in the art for example byGarnett and Baldwin, Cancer Research 1986 46:2407-2412. The use oftoxins conjugated to monoclonal antibodies for the therapy of variouscancers has also been described by Pastan et al. Cell 1986 47:641-648.Yttrium-90 labeled monoclonal antibodies have been described formaximization of dose delivered to the tumor while limiting toxicity tonormal tissues (Goodwin and Meares Cancer Supplement 1997 80:2675-2680).Other cytotoxic radionuclides including, but not limited to Copper-67,Iodine-131 and Rhenium-186 can also be used for labeling of antibodiesagainst CC2.

Antibodies which can be used in these in vivo methods includepolyclonal, monoclonal and omniclonal antibodies and antibodies preparedvia molecular biology techniques. Antibody fragments and aptamers andsingle-stranded oligonucleotides such as those derived from an in vitroevolution protocol referred to as SELEX and well known to those skilledin the art can also be used.

The present invention is further described by the following examples.These examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain aspects of the invention, do not portray thelimitations or circumscribe the scope of the disclosed invention.

EXAMPLES

The examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing example can be carried out as described in standard laboratorymanuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

Real-Time quantitative PCR with fluorescent Taqman probes is aquantitation detection system utilizing the 5′-3′ nuclease activity ofTaq DNA polymerase. The method uses an internal fluorescentoligonucleotide probe (Taqman) labeled with a 5′ reporter dye and adownstream 3′ quencher dye. During PCR, the 5′- 3′ nuclease activity ofTaq DNA polymerase releases the reporter, whose fluorescence can then bedetected by the laser detector of the Model 7700 Sequence DetectionSystem (PE Applied Biosystems, Foster City, Calif., USA).

Amplification of an endogenous control was used to standardize theamount of sample RNA added to the reaction and normalize for ReverseTranscriptase (RT) efficiency. Either cyclophilin,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA(rRNA) was used as this endogenous control. To calculate relativequantitation between all the samples studied, the target RNA levels forone sample were used as the basis for comparative results (calibrator).Quantitation relative to the calibrator is obtained using the standardcurve method or the comparative method (User Bulletin #2: ABI PRISM 7700Sequence Detection System).

To evaluate the tissue distribution, and the level of CC2 in normal andtumor tissue, total RNA was extracted from normal tissues, tumortissues, and from tumors and the corresponding matched normal tissues.Subsequently, first strand cDNA was prepared with reverse transcriptaseand the polymerase chain reaction was done using primers and Taqmanprobe specific to CC2. The results were analyzed using the ABI PRISM7700 Sequence Detector and are provided in the following table. Theabsolute numbers are relative-levels of expression of CC2 compared tothe kidney (calibrator).

The absolute numbers depicted in Table 1 are relative levels ofexpression of CC2 in 12 normal different tissues. All the values arecompared to normal kidney (calibrator) These RNA samples arecommercially available pools, originated by pooling samples of aparticular tissue from different individuals.

TABLE 1 Relative Levels of CC2 Expression in Pooled Samples TissueNORMAL Colon-Ascending 536 Endometrium 0 Kidney 1 Liver 10 Ovary 4Pancreas 22 Prostate 332 Small Intestine 2539 Spleen 0.0 Stomach 2062Testis 112 Uterus 2

The relative levels of expression in Table 1 show that the higher levelof expression of CC2 mRNA is in tissues from the gastrointestinal tract,small intestine (2539), stomach (2062), and colon (536), with a lowerlevel of expression in prostate (332), and testis (112). These resultsestablish that CC2 mRNA expression is highly specific-forgastrointestinal tissues including not only the colon but also the smallintestine and stomach.

The absolute numbers in Table 1 were obtained analyzing pools of samplesof a particular tissue from different individuals. They should not becompared to the absolute numbers originated from RNA obtained fromtissue samples of single individuals depicted in Table 2.

The absolute numbers depicted in Table 2 are relative levels ofexpression of CC2 in 78 pairs of matching samples. All the values arecompared to normal kidney (calibrator). A matching pair is formed bymRNA from the cancer sample for a particular tissue and mRNA from thenormal adjacent sample for that same tissue from the shame individual.

TABLE 2 Relative Levels of CC2 Expression in Pooled Samples NormalCancer Adjacent Sample ID Tissue Tissue Tissue StoAC93 Stomach 1 64860279026 Sto728 Stomach 2 0 40 Sto758S Stomach 3 21029 2903 Sto915SStomach 4 3488 56 StoAC99 Stomach 5 1162 330 Sto115S Stomach 6 404 146Sto15S Stomach 7 4636 14 Sto17S Stomach 8 59662 538 Sto261S Stomach 953061 8977 Sto264S Stomach 10 27492 84643 Sto27S Stomach 11 20784 61Sto288S Stomach 12 0 67 Sto531S Stomach 13 53192 8847 Sto539S Stomach 141492 27 Sto542S Stomach 15 26382 425 Sto610S Stomach 16 1029 20 Sto88SStomach 17 3846 12 StoAc44 Stomach 18 1.7 78 StoMT54 Stomach 19 971 67StoTA73 Stomach 20 35653 6020 SmI21XA Small Intestine 1 31016 10022SmIH89 Small Intestine 2 645 2227 ClnB56 Colon-Cecum 1 6816 971 ClnAS45Colon-Ascending 2 8757 5501 ClnCM67 Colon-Cecum 3 2394 578 ClnAS67Colon-Ascending 4 1566 1198 ClnAS43 Colon-Ascending 5 127934 923 ClnAS46Colon-Ascending 6 96620 3316 ClnAS98 Colon Ascending 7 83822 392 ClnAS89Colon-Ascending 8 10231 4 ClnTX01 Colon-Transverse 9 92 331 ClnTX89Colon-Transverse 10 11114 17 ClnTX67 Colon-Transverse 11 683 189 ClnMT38Colon-Splenic 0 6230 flexture 12 ClnSG89 Colon-Sigmoid 13 2557 1243ClnSG67 Colon-Sigmoid 14 39 132 ClnSG33 Colon-Sigmoid 15 17080 118542ClnSG45 Colon-Sigmoid 16 243 80 ClnB34 Colon-Rectosigmoid 17 130 11ClnCXGA Colon-Rectum 18 790 47152 ClnRC67 Colon-Rectum 19 724 419ClnC9XR Colon-Rectosigmoid 20 425 113 ClnRS45 Colon-Rectosigmoid 2142202 1117 ClnRC01 Colon-Rectum 22 2693 99 ClnRC89 Colon-Rectum 23 02402 Bld46XK Bladder 1 0 0 Bld66X Bladder 2 15 4 Bld32XK Bladder 3 8.50.4 Kid126XD Kidney 1 5 5 Kid12XD Kidney 2 2 0 Kid5XD Kidney 3 3.7 0.8Kid6XD Kidney 4 4.3 0 Kid106XD Kidney 5 0 0.8 Liv42X Liver 1 2 1 Liv15XALiver 2 0.2 0.7 Liv94XA Liver 3 0 1.4 LngAC69 Lung 1 2 0 LngBR94 Lung 23 0 Lng47XQ Lung 3 0 0 Mam59X Mammary Gland 1 0 0 MamB011X Mammary Gland2 0 0 MamA06X Mammary Gland 3 15 20 Ovr103X Ovary 1 4 0 Ovr130X Ovary 23 3 Pan71XL Pancreas 1 69458 15147 Pan82XP Pancreas 2 0 0 Pan77XPancreas 3 0 0 Pan92X Pancreas 4 4696 52 PanC044 Pancreas 5 34 0 Pro12BProstate 1 21 2 Pro23B Prostate 2 23 6 Pro13XB Prostate 3 6 23 Pro34BProstate 4 152 75 Pro20XB Prostate 5 112 13 Pro65XB Prostate 6 60 683Tst39X Testis 1 2361 17 Endo10479 Endometrium 1 32 0 Utr85XU Uterus 1 00 0 = Negative

In the analysis of matching samples, the higher levels of expression forCC2 are in stomach, small intestine, and colon. This pattern shows ahigh degree of specificity for gastrointestinal tissues including, notonly the colon, but also the stomach and small intestine. These resultsconfirm the tissue specificity results obtained with the panel of normalpooled samples (shown in Table 1).

The level of mRNA expression in cancer samples and the isogenic normaladjacent tissue from the same individual were also compared. Thiscomparison provides an indication of specificity for the cancer stage(e.g. different levels of mRNA expression in the cancer sample comparedto the normal adjacent tissue). Table 2 shows overexpression of CC2 in15 primary stomach cancer tissues compared with their respective normaladjacent (stomach samples #3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17,19, and 20). There is overexpression in the cancer tissues for 75% ofthe stomach matching samples tested (total of 20 stomach matchingsamples).

CC2 is also differentially expressed in the two tested matching samplesfor cancer of the small intestine. Sample #1 shows upregulation for themRNA of CC2 in cancer, whereas sample #2, shows lower expression incancer.

CC2 is differentially expressed in twenty-three matching samples forcolon cancer. Samples #1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 16, 17, 19,20, 21 and 22 show upregulation for the mRNA of CC2 in cancer, whereassamples #9, 12, 14, 15, 18, and 23 show lower expression in the cancersample when compared to the normal adjacent tissue.

Altogether, the high level of tissue specificity for gastrointestinaltissues, plus the mRNA differential expression in several of the primarystomach, small intestine, and colon matching samples tested indicate CC2to be a diagnostic marker for gastrointestinal cancers including notonly colon cancer, but also stomach cancer and cancer of the smallintestine.

1. A method for detecting the presence of cancer of the stomach or smallintestine in a patient comprising: (a) measuring levels of CC2 in cells,tissues or bodily fluids from a patient; and (b) comparing the measuredlevels of CC2 with levels of CC2 in cells, tissues or bodily fluids froma normal human control sample of the same, type of cells, tissues orbodily fluids as the patient, wherein an increase in measured levels ofCC2 in said patient versus normal human control sample of the same typeof cells, tissues or bodily fluids as the patient is associated with thepresence of cancer of the stomach or small intestine, wherein CC2comprises SEQ ID NO:1 or SEQ ID NO:2.
 2. The method of claim 1 whereinthe CC2 comprises SEQ ID NO:1.
 3. The method of claim 1 wherein the CC2comprises SEQ ID NO:2.
 4. The method of claim 1 wherein CC2 levels aremeasured in cells from the patient and normal matched human controlsample.
 5. The method of claim 1 wherein CC2 levels are measured intissue from the patient and normal matched human control sample.
 6. Themethod of claim 1 wherein CC2 levels are measured in bodily fluid fromthe patient and normal matched human control sample.